The antigen-antibody reaction is the basis for all immunological test methods. Certain proteins known as antibodies are produced by mammals in response to the presence of an antigen, that is a foreign substance, which can be another protein or a carbohydrate. This normal body response to a foreign substance has led to the development of a number of techniques which are used to diagnose various diseases, disorders and physiological conditions. In a general sense, the component of the antibody-antigen reaction to be detected is defined herein as the immune species while the other corresponding component is considered the receptor.
In vitro tests for the presence of a suspected protein, antigen or antibody in a biological sample are carried out by adding the immunological counterpart to the biological sample. If the suspected substance is present, the resulting antigen-antibody reaction can be demonstrated by precipitation of the antigen-antibody complex. This reaction complex is generally difficult to detect visually. For this reason, either antibodies or antigens are often bound to insoluble particles, for example polymer latex particles, so that when the complex is formed, it is readily detectable from the resulting agglutination either by observing the presence of clumping or a detectable tracer associated with the particles. Agglutination then is characterized by the clumping of particles from a suspension of particles. Further details of known agglutination methods are provided in U.S. Pat No. 4,419,453 (issued Dec. 6, 1983 to Dorman et al) and U.S. Pat. No. 4,459,361 (issued July 10, 1984 to Gefter).
Of the several groups of Streptococci, group A Streptococcus (S. pyogenes) is primarily responsible for causing pathological conditions in humans, such as B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac sequelae, glomerulonephritis, septic sore throat and puerperal sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence in an early stage of infection so that an appropriate course of treatment may be selected. Early tests for detection required culturing a biological sample for long periods of time, usually at least 18 and up to 48 hours. In most cases, such lengthy tests delay treatment making them undesirable.
More recent tests for Streptococcus A have been described which are allegedly quicker than the culturing techniques. U.S. Pat. No. 4,618,576 (issued Oct. 21, 1986 to Rosenstein et al) describes an agglutination test using certain enzymes to extract the antigen directly from the swab used to obtain a specimen from the throat. A kit comprising an applicator means for collecting the specimen, an extraction reagent containing the enzymes and suitable indicator reagents is also described. The described method comprises placing the extracted antigen in the wells of a sample plate along with latex particles coated with antibodies. After agitating the wells mechanically to facilitate antigen-antibody reaction, any agglutination is observed in the mixture in the wells. This method is disadvantageous because the agglutinate is not readily observable except with a microscope, and requires extraction enzymes prepared from a bacterium which must be cultured.
Other agglutination assays using various latex particles and coloring techniques for observing the agglutinate are described in E.P. Publications No. 150,567 and No. 174,195. U.S. Pat. No. 4,552,839 (issued Nov. 12, 1985 to Gould et al) describes an agglutination assay carried out by concentrating particles in a small area on a solid surface. The concentrated particles can have antibodies or label attached thereto to aid in detection of the analyte, that is, the antigen. The conditions of the assay are chosen to adjust the reaction of beads to each other. For example, the ionic strength of the aqueous medium is adjusted based on the natural charge of the particles in order to maximize agglutination. Generally, the ionic strength is varied from about 0.0001 to about 0.1. Once the agglutinate is formed and concentration on a bibulous material (for example, filter paper) is effected, a phosphate buffered saline wash containing a surfactant is applied to aid in wicking and separating unbound materials from bound materials (Col. 6, lines 50-55).
Current agglutination assays for a number of multivalent immune species [for example, Streptococcus A, human chorionic gonadotropin (hCG), Chlamydia, Gonorrhea, herpes, HIV, or human immunodeficiency viruses (formerly known as LAV or HTLV viruses) and others] are limited in usefulness in several respects. They are generally difficult to interpret, non-quantitative, subject to interferences and often insensitive. In order to improve the sensitivity and accuracy of agglutination assays for multivalent immune species carried out using antibody-bound particles, it has been found that a means is needed for keeping agglutinated materials from breaking apart and, at the same time, effectively separating agglutinated and unagglutinated materials.